Description - Integral plasma membrane proteins are internalized for degradation or recycling via the endocytic pathway. Internalized plasma membrane proteins are sorted for degradation or recycling at the Multivesicular Body (MVB) prior to vesicle formation and internalization. Genes involved in protein trafficking to the vacuole in S. cerevisiae are categorized into six classes (A-F) based on vacuole morphology in mutant strains. Many of the class E Vps proteins are required for protein sorting at the MVB and are conserved between yeast and humans. SnfSp is a high affinity plasma membrane glucose sensor that detects extracellular glucose. Upon glucose binding, SnfSp activates a signal transduction pathway resulting in transcriptional activation of hexose transporters that internalize glucose for metabolic processing. The mechanism of glucose sensing is similar between yeast and humans. We have generated evidence that endocytosis of SnfSp likely plays a key role in regulating glucose sensing in yeast. The experiments outlined in this proposal aim to understand how the endocytic pathway regulates glucose sensing and to identify novel components of vesicle formation at the MVB. The first aim utilizes Snf3-GFP localization and a pathway readout assay in class E vps mutant strains with changing glucose conditions to further understand how the endocytic pathway regulates glucose sensing via SnfSp. A novel genetic screen will be performed in the second aim to uncover factors required for membrane invagination at the MVB. These factors will be used to further investigate MVB vesicle formation and the role of the endocytic pathway in glucose sensing. This proposal will have an impact on several human health issues because HIV viral particle formation and viral budding requires the mammalian class E Vps proteins. Also, both S. cerevisiae and cancerous tumors metabolize glucose by fermentation in the presence of oxygen. This unique aspect of tumor biology has been utilized for diagnostic purposes and has recently been the focus of targeted therapeutics. Therefore, the discoveries made here will aid our understanding of viral proliferation and tumor biology. Relevance - Cancer and HIV are two major public health issues. This proposal describes a basic science approach to understanding more about tumor biology and viral proliferation using yeast as a model organism. [unreadable] [unreadable] [unreadable]